The present invention discloses a novel, commercially viable process for extraction of bioactive, high-purity (>70%) furostanolic saponins, particularly protodioscin (>30%) from fenugreek seeds. Protodioscin has Molecular Formula C51H84O22, CAS No. 55056-80-9 and structure as given in FIG. 1.
Plants produce a vast and diverse assortment of organic compounds, the great majority of which do not appear to participate directly in their growth and development. These substances, traditionally referred to as secondary metabolites or plant natural products, often are distributed among limited taxonomic groups within the plant kingdom. The functions of secondary metabolites remain largely unknown, although a number of compounds have been associated with attributes useful to the plants e.g. protection against herbivores and protection against microbial infection, as attractants for pollinators and seed-dispersing animals, and as compounds that influence competition among plant species (allelochemicals). There is a growing interest in plant natural products, since these products often have a wide range of applications in different kinds of industries, including pharmaceutical industries, cosmetic industries, food industries, detergent industries, etc.
Secondary Metabolites of Commercial Interest—Saponins
Saponins are an example of a group of plant secondary metabolites. Saponins are glycosylated compounds classified as either triterpenoids, steroids, or steroidal glycoalkaloids. Saponins consist of one or two sugar moieties which are coupled to the aglycon (mono- and bisdesmosides, respecitvely). Saponins can be hydrolysed to sapogenins and sugar moieties by acid hydrolysis or enzymatic methods. Saponins are water soluble high molecular weight compounds with molecular weights ranging from 600 to more than 2,000 daltons.
Definition: In simple terms, saponins can be defined as “molecular complexes consisting of any aglycone (sapogenin) attached to one or more sugar chains.”
Saponins thus describes a class of natural products which are structurally constructed of aglycone (triterpene or steroidal) and sugars (pentose(s), hexose(s), and/or uronic acid(s). Accordingly, Saponins can be classified according to their aglycone composition as follows:    1) Triterpene glycosides    2) Steroid glycosides    3) Steroid alkaloid glycosides
The Furostanolic saponins of the present invention fall in categories 2 and 3. The asymmetric distribution of their hydrophobic (aglycone) and hydrophilic (sugar) moieties confers an amphipathic character to these compounds which are largely responsible for their detergent-like properties. In some cases saponins may be acylated with organic acids such as acetic, malonic, angelic and others as part of their structure (Hostettmann K. and Marston A. Saponins, Cambridge University Press, Combridge. 1995.; Rouhi A M., Chem. Eng. News 73(37):28-35, 1995.; Leung A Y., and Foster S., Encyclopedia of Common Natural Ingredients Used in Food, Drugs, and Cosmetics, 2.sup.nd ed., John Wiley and Sons (Wiley-Interscience), New York (1996).
Saponins—Applications and Commercial Importance
Saponins have significant commercial uses. Until recently, saponins have been largely overlooked except in cases where their presence was considered toxic. There are human benefits to the use of galactomannans as well. In particular, it has been reported that fenugreek galactomannan is most evident in the lowering of cholesterol in both liver and blood plasma.
Cosmetic and detergent industries: Saponins have the ability of lowering surface tension and the word “saponin” also reflects this as “sapo” is the latin word for soap. Saponins therefore have potential applications in the cosmetic and in the detergent industries.
Pharmaceutical and therapeutic applications: Saponins furthermore have the ability of forming insoluble complexes with cholesterol, which makes some of them suitable for use in the pharmaceutical industry as cholesterol lowering agents. Saponins also have other therapeutical effects. Saponins from chestnut do e.g. possess anti-inflammatory characteristics. Saponins, when injected into the blood stream, are furthermore highly toxic due to their haemolytic properties. Saponins are usually relatively harmless when ingested orally. Steroidal saponins are of great interest owing to their relationship with such compounds as the sex hormones, cortisone, diuretic steroids, vitamin D and cardiac glycosides. Also, saponins are associated with formation of immunostimulating complexes (ISCOMs) (Morein et al., (1995) Clinical Immunotherapeutics 3: 461-475) that are useful in vaccine strategies.
Food and nutraceuticals industry: Saponins have flavoring, sweetening, antioxidant, foaming, complexing, sequestration, anticarcinogenic and antimicrobial properties. They could be used as an antioxidant, emulsifier, surfactant, ingredient for nutraceutical in food, health food and nutraceuticals industry and as such represent another value-added fraction for recovery.
Limitations to Widespread Use of Saponins
Cost: At present however, a major obstacle in exploiting the wide range of potential applications of saponins is the fact that commercially available saponins are relatively expensive. Commercially available plant extracts containing saponins are extracts of Saponaria officinalis, Quillaia bark and stem, Castanea sativa seeds, and extracts of various Yucca species. Liquorice root, primula root, and senega root can also serve as raw material for saponin extracts.
Limited raw material sources: A problem in this field is that the available sources of saponin extracts are relatively few. And in some cases, e.g. Quillaia bark, the plants are often sparse and expensive because they cannot be cultivated in an efficient manner. Usually the saponins are present in relatively low concentrations. The commercially available saponin extracts are thus often expensive and/or sparse.
Complex structures, which limit chemical synthesis: It should also be noted that saponins are quite complicated compounds and it has not so far been profitable to develop methods for chemical synthesis of these compounds.
Plant extracts containing saponins and sapogenins are thus of general interest within a wide range of different industries. There is therefore a growing need in the art for alternative sources of saponin extracts and these plant sources should preferably be cheap, easy to obtain, and preferably the saponin content should be relatively high. For the general description of saponins, extraction, production, and use of saponins reference is made to Ullman's Encyclopedia of Industrial Chemistry (1993), Vol. A23, pp. 485-498. Furthermore, the literature is abundant concerning the extraction, composition and specific effects of the individual saponins derived from plant materials.
Extraction of Saponins
Processes for extraction of commercially useful saponins and also bioactive saponins useful for pharmaceutical applications, from various plant sources have been described in prior art patents. However, saponins of prior art are quite different from the saponins of the present invention which are specifically ‘furostanolic saponins’. None of the processes in prior art disclose a method for obtaining high-purity (>70%), bioactive fractions rich in furostanolic saponins particularly protodioscin (>30%) as has been disclosed in the present invention. The process is quite efficient and economical, resulting in yield of 2-4% of the purified bioactive fractions.
Processes for extraction of saponins as described in the prior art, from the major source, soybeans and other plant sources are discussed below. This is followed by discussion on processes for extraction of saponins specifically from sources which are regarded as rich sources of furostanolic saponins viz Tribulus terrestris, Dioscorea deltoidea and Fenugreek seeds. The present invention discloses a novel method for the extraction of high-purity (>70%) bioactive fractions rich in furostanolic saponins, particularly protodioscin from fenugreek seeds.
Extraction of Saponins from Soybeans: Processes for extraction of saponins from soybeans have been described in US patent applications no 20040013791, 20040101579, 20050123662 and 20050037099. These processes are quite different from the novel process of the present invention which is for extraction of a particular class of saponins i.e. furostanolic saponins, particularly protodioscin from fenugreek seeds, at high levels of >70% in the extracts. Moreover, quite a few processes utilize strong acidic or alkaline conditions for separation of the desirable saponins, whereas process of the present invention is a completely eco-friendly process, not utilizing any strong acids or alkalis. The entire process is carried out at or near neutral pH i.e. between pH 6 to 8.
It is known that soya contains saponin and isoflavone components in addition to saccharide and amino acid components, as well as proteins and mineral salts in amounts which depend on their geographical origin and the conditions under which the plant was cultivated and harvested. Soybean saponins are a generic name of saponins contained in starting soybeans and are present in concentrations of about 2 to 4% by weight. Saponins are classified into group A saponin, i.e., bisdesmoside saponin, wherein an aglycone skeleton is “soyasapogenol A” and sugar chains are attached to C-3 position and C-22 position of the aglycone through ether bonds; group B saponin, i.e., monodesmoside saponin, wherein an aglycone skeleton is “soyasapogenol B” and a sugar chain is attached to C-3 position of the aglycone through ether bond; and the like. In addition, saponin E, wherein a moiety of a sugar chain is acetylated is also reported (Kitagawa et al, Chem. Phrm. Bull, 33, (1985). Thus, saponin contents have been divided into three classes depending on the chemical structure of their triterpene components: soya saponins of groups A, B and E (Okubo K. et al., ACS Symp., Ser. 546, 330, 1994). 1 1 R.sub.1 R.sub.2 R.sub.3 Group A; Saccharide Saccharide OH chain chain Group B Saccharide OH H chain Group E Saccharide —OH chain.
Isoflavone components consist of glucoside isoflavones (daidzin, genistin and glycitin) which can contain acyl radicals, e.g. malonyl radicals, linked to the saccharide chain. 2 2 R.sub.1 R.sub.2 R.sub.3 Daidzin H H D-Glucose Glycitin OCH3 H D-Glucose Genistin H OH D-Glucose Daizdein H H H Glycitein OCH.sub.3 H H Genistein H OH H
US patent application no 20040013791 discloses a soy protein material having a very low isoflavones content and a high content of saponins. The process for producing the low isoflavones, high saponins soy protein material involves removing fiber from a defatted soybean material and obtaining a liquor that is subsequently pasteurized. Next, sugars and other small molecular weight components are optionally removed from the liquor using membrane separation to increase the protein content of the final material. The resulting liquor or retentate is subjected to adsorptive removal of isoflavones, and is optionally pasteurized and spray dried. The process thus involves pasteurization and membrane filtration steps, which are not used in the process of present invention at all. Also, there is no disclosure about any process for obtaining high-purity (>70%) bioactive fractions rich in furostanolic saponins, particularly protodioscin as has been disclosed in the present invention.
US patent application no 20040101579 relates to novel extracts obtained by extraction of ripe complete soya beans or from oil-free soya flour (Glycine max (L.) MERRIL, Leguminosae family), their production and formulations containing these extracts. The novel extracts are characterized by their content of isoflavones and saponins in defined ratios. The extracts are characterized by a content of glucoside isoflavones of at least 13% by weight and a content of 0.6 to 1.5 parts by weight of group 3 soya saponins and per 1 part by weight of glucoside isoflavones. The extract is mainly for treating pre- or post-menopausal symptoms in a female subject and also breast cancer in females. The application does not describe any process for obtaining fractions rich in furostanolic saponins (>70%), particularly protodioscin as has been disclosed in the present invention. Also, the present invention discloses furostanolic saponin rich fractions, particularly protodioscin, to enhance testosterone production and not for treatment of menopausal symptoms or cancer.
United States Patent Application no. 20050123662 describes a simple and novel process for obtaining highly pure saponins at a high yield, on an industrial scale from soybeans, starting from isoflavones extracted from soybeans. The same is discussed in detail as follows:
Conventionally, in order to obtain a material containing saponins at a high concentration from starting soybeans such as whole soybeans, defatted soybeans, soybean hypocotyls, and the like, it was known that appropriate separation of saponins from isoflavones is important. A general scheme in the conventional process consisted of first stage production steps involving extraction with a solvent such as a lower alcohol or acetone; adsorption of the resultant extract with a synthetic adsorption resin such as HP-20 and XAD-2 or activated carbon; washing the absorbed extract with a low concentration of a lower alcohol (10 to 40% by volume) and purification by gel filtration such as LH-20, or the like, or partition chromatography after carrying out and then elution of a fraction containing saponins with a high concentration of a lower alcohol (70 to 80% by volume).
However, in order to obtain a highly pure saponin fraction in the above-described processes, the final purifying step by means of gel filtration or partition chromatography was required and led to a low yield of saponins to about 1 to 2%. This was problematic from the viewpoints of facility costs and production efficiency for industrially putting such a process into practice. On the other hand, in order to extract saponins with a solvent at a high yield, the extraction was conventionally being carried out at a high temperature. However, even when such extraction was carried out, material containing saponins which satisfied requirements of high purity and high yield without complicated operation, could not be obtained.
To enhance the yield of saponins, raising temperature during extraction or altering pH are well known conventional approaches which were utilized by the inventors. However, it was found that though yield of saponins could be improved under high temperature conditions, but at the same time solid materials other than saponins also got extracted, which conversely caused a decrease in purity of saponins. For purification, two complicated stages further were required, which though resulting in pure saponin fractions, resulted in poor yields.
The inventors observed that hydrophobicities of saponins are particularly close to those of isoflavone glycoside, acetyl isoflavone glycoside, and isoflavone aglycone, among isoflavones. Therefore, it is difficult to separate them by utilizing these properties. Among isoflavones, malonyl isoflavone glycoside has a dissociation group and thus has a relatively high polarity. Therefore, malonyl isoflavone glycoside can be easily separated from saponins with an adsorbent. In a natural state, soybeans contain a largest amount of malonyl isoflavone glycoside. However, malonyl isoflavone glycoside is unstable to heat and is easily converted into isoflavone glycoside, acetyl isoflavone glycoside, or isoflavone aglycone by heating, which is typically employed in conventional processes. Once these compounds were generated as a result of heating, it became very difficult to separate these from saponins Hence, if heating could be avoided an multistage extractions carried out, relatively pure saponin fractions at high yields could be obtained. The same was tried and found to be successful by the inventors of the above prior art application. When multi-stage extraction was carried out under mild conditions at which conversion of malonyl isoflavone glycoside in starting soybeans into isoflavone glycoside, acetyl isoflavone glycoside, or isoflavone aglycone was avoided, it was found that saponins could be separated from isoflavones extremely efficiently only by subsequent one-stage purification step, i.e., treatment with an adsorbent to obtain highly pure saponins at a high yield. Thus, a simple and easy process for producing a saponin-containing material having an extremely high purity at a high yield on an industrial scale was standardized.
A comparison of the steps involved in the process of the above prior art document and the process as described in the present invention shows that the processes are entirely different, though both are aimed at extraction of saponins. The process of the prior art document consists of one stage purification whereas process of the present invention is a multi-step purification. Secondly, process of the prior art is directed towards saponin extraction from soybeans, whereas process of the present invention is directed toward the extraction of specifically furostanolic saponins rich in protodioscin from fenugreek seeds. As the nature and content of the contaminating compounds i.e. isoflvanones and also nature of the saponins itself is different in both cases, the extraction processes in both cases are different.
Moreover, the saponins extracts of the prior art document as discussed above, are intended for a variety of physiological activities such as anti-obesity activity, antioxidant activity, and an immunity activation activity. Then, when a material containing saponins at a high concentration is available, the material can be utilized by simply adding it to various foods and drinks. In contrast, the saponins of the present invention are specifically furostanolic saponins, particularly protodioscin intended for elevating testosterone levels and promoting anabolism and to be consumed in well-defined dosage forms e.g. tablets or capsules.
United States Patent Application no. 20050037099 describes a simple and inexpensive semi-industrial technology for isolation and purification of active plant compounds, including isoflavones, sitosterols, saponins (sapogenins), and phospholipids for topical application. Soybeans are particularly suitable as a plant source; however, any other isoflavone-containing plants (e.g., red clover and kudzu) can be used as a source of active compounds, in the process of the described invention. Molasses, enriched fluor or any other non-protein concentrate can be used as a source material for the isolation and purification process. The composition of the above invention is suitable for topical application for preventing and treating changes associated with skin aging and formation of abnormal skin lesions. The composition is derived by hydrolyzing a source composition in an acidic solution. The source composition can be, e.g., a soy composition and can include one or more isoflavones, phytoestrogens, sitosterols and saponins. After hydrolysis, water is added to the acidic solution to produce a first precipitate, which is then dissolved in an alcohol solution. The alcohol solution is then separated from undissolved portions of the first precipitate, and the alcohol in the separated alcohol solution is eliminated to produce a second precipitate of solids that were dissolved in the alcohol. This second precipitate is then dissolved in an organic solvent to produce a composition suitable for inclusion in a dermatological or cosmetic preparation.
Thus, the composition described is a mixture of various phytocompounds and the extraction process is also not specific for saponins alone. Moreover, it involves use of acids to produce a first precipitate and does not involve any chromatographic purification steps. In contrast, the present invention is specifically directed towards an extraction process for obtaining high-purity (>70%) fractions rich in furostanolic saponins, particularly protodioscin, from fenugreek seeds. It does not involve use of any strong acids at all. All steps are carried out at primarily neutral pH i.e. 6 to 8. Also, the purification process of the present invention is a multi-step process involving chromatographic purification also.
Extraction of Saponins from Other Plant Sources:
                1. Anemone Raddeana         2. Edible beans,        3. Plants of the family Myrsinaceae,        4. Quillaja saponaria Molina (soap tree)        5. Sapindus trifoliatus         6. Wenguanguo or Xanthoceras sorbifolia         7. Plants from the genus Panax (Panax ginseng, Panax quinquefolium and Panax notoginseng)        8. Honeysuckle (Lonicera japonica Thunb.)        9. Plant species, notably from the genera Smilax, Asparagus, Anemarrhena, Yucca and Agave         10. Xanthoceras sorbifolia         11. Butter tree of the Sapotaceae family        
1. Anemone Raddeana: US patent application no 20040067263 discloses a process for preparation of a crude saponin rich extract from rhizome, stem, leaf, flower, and fruit of Anemone Raddeana. The same is useful for treating malignant neoplasm diseases. The bioactive saponins disclosed are thus quite different from that of the present invention, which specifically relates to extraction of high purity (>70%), bioactive furostanolic saponins, which enhance testosterone production.
2. Edible beans: United States Patent Application no. 20040131749 relates to a composition comprising a phytochemical, in particular, polyphenolic extract derived from edible beans and containing one or more of the following: anthocyanins, flavonols, proanthocyanindins, isoflavones, lectins, saponins, sapogenins, vitamins, minerals and functional proteins. The invention further provides a process for the production of an anthocyanin or flavonol extract from an edible bean source, the process comprising providing the edible beans, contacting the edible beans with an aqueous solution to produce an aqueous extract, and separating the aqueous extract from the edible beans. The present invention relates, in general, to a process of isolating phytochemical, in particular polyphenolic compounds from commercial edible bean processing waste streams for use as natural colors and functional foods and extracts obtained by such process. These compounds also have potential for use as nutraceutical ingredients in the following health related areas: antioxidants, cardiovascular disease protection, anti-diabetic effects, anti-inflammatory effects, antiviral properties, anti-tumorgenic properties, improvement of night vision, anti-inflammatory activity and anti-stress. The resulting extracts can be formulated for treatment of these conditions. Further utilization can occur for use as cosmetic ingredients and natural colors. The invention also relates to novel compositions comprising extracts prepared by the process of the invention. Though saponins are present as components in the polyphenolic extracts of this invention, they are neither the main ingredients nor of the same nature or biological action as the saponins of the present invention, which are specifically bioactive, high-purity (>70%), furostanolic saponins, particularly protodioscin. Moreover, the intended biological use of the bioactive saponins in both is entirely different—the former proposing use of the polyphenol extracts, containing saponins for a wide variety of physiological processes while the present invention specifically pertains to furostanolic saponins, particularly protodioscin, for enhancing testosterone levels and anabolic processes.
3. Plants of the family Myrsinaceae: United States Patent Application no. 20040138151 discloses antiprotozoal saponins isolated from plants of the family Myrsinaceae and used to decrease the infectiousness of and reduce the mortality associated with protozoan parasites of the genus Leishmania which are responsible for a group of conditions known as leishmaniases. The invention relates to one or more triterpene saponins obtainable by the processes described for extraction of the compounds, whether as a mixture or as isolated products. The saponins described i.e. triterpene saponins and their methods of extraction are quite different from the present invention which refers specifically to extraction of only one specific category of saponins ie. high-purity (>70%) furostanolic saponins, particularly protodioscin, from fenugreek seeds.
4. Quillaja saponaria Molina (soap tree): United States Patent Application no. 20050175623 discloses compositions comprising saponins, extracted from Quillaja saponaria Molina (soap tree), which are used as therapeutic compounds for the treatment and prevention of cancer diseases or as a dietary supplement that offers tumor cell killing and tumor cell inhibition, and also as a anticancer potentiator with other anticancer agents. The saponin derived novel anticancer agents, include both triterpenoid and steroidal saponins found in the bark of Quillaja saponaria Molina (soap tree). The Saponins include but are not limited to, sapogenins, and its prosapogenins with one or more sugar moieties. The method of extraction of saponins has not been disclosed as the same were purchased from commercial sources as ‘ready-to-use’ extracts. Invention is thus directed towards novel uses of saponin extracts, rather than disclosing a process of extraction of furostanolic saponins as has been described in the present invention.
5. Sapindus trifoliatus: United States Patent Application no. 20050249831 discloses a process for a herbal extract comprising a mixture of saponins obtained from sapindus trifoliatus (Common name, Ritha) for anticonvulsant activity. The pericarp of the fruit of the plant, which constitutes 62% of the fruit contains, glucose, saponins and primary metabolites. The saponins present in the fruit on acidic hydrolysis give the triterpenoid hederagenin, D-glucose, L-rhamnose and D-xylose and Arabinose. [The Wealth of India, Vol IX, CSIR Publication, by NISCOM, New Delhi, India, 1998, pp 227-29]. The extract is prepared from the pericarp of the fruit of S. trifoliatus, and compres from 4 to 8% w/w of hederagenin as the bioactive component and ii. pharmaceutically acceptable additives. The process comprises the steps of extraction of the pericarp of the fruit of S. trifoliatus with water or an alcohol or a mixture thereof at ambient to boiling temperature for 0.5 to 24 hours, b. lyophilization of the aqueous, alcoholic or aqueous alcoholic extract containing a mixture of saponins to give a lyophilized powder, containing a mixture of saponins, and c. reconstitution of the lyophilized extract in water to achieve a concentration of hederagenin between 0.001 to 1.0 (% w/v). The invention discloses the preparation of bioactive pure compounds from a mixture of triterpenoid saponins extracted from the pericarp of S. trifoliatus. 
However, the present invention is directed specifically towards the extraction of furostanolic saponins, which are a separate category of saponins from the triterpenoid saponins as detailed in the above patent. The extraction process of the present invention is altogether different and results in furostanolic saponin rich fractions (>70%) and not extraction of any triterpenoid saponins.
6. Wenguanguo or Xanthoceras sorbifolia: United States Patent Application no. 20050276872 discloses the extraction of saponins and other constituents including alkaloids, coumarins, saccharides, proteins, polysaccharides, glycosides, tannins, acid, flavonoids and others from husks or fruit-stems or seed shells or leaves or branches or stems or kernels or roots or barks of Wenguanguo or Xanthoceras sorbifolia. The category of saponins is oleanene triterpenoidal saponin with the characteristics that at least one angeloyl group is attached to Carbon 21 or/and 22, or/and linked to the sugar. The compounds of the present invention have various pharmaceutical and therapeutic applications. In contrast, the saponins of the present invention are not triterpenoid saponins, but high-purity (>70%) steroid saponins (furostanolic saponins) extracted from fenugreek seeds.
7. Plants from the genus Panax (Panax ginseng, Panax quinquefolium and Panax notoginseng): United States Patent Application 20060013897 discloses compositions for cancer therapy comprising two or more saponins and/or sapogenins. Ginseng has long been recognized as a general tonic and a benign and safe herb. Many of the components of ginseng have been isolated and have been classified as: ginsenosides, carbohydrates, nitrogenous compounds, fat-soluble compounds, vitamins and minerals. The saponins derived from ginseng (also called “ginsenosides”) are believed to be the main active components of ginseng. Saponins, in general, are composed of a sugar portion (glycon) and a non-sugar portion (aglycon or sapogenin). The sapogenins in ginseng, the backbone of saponins, are further classified into three types: protopanaxadiol and protopanaxatriol (which are tetracyclic terpenoids of the dammarane series), and oleanoic acid. More than dammarane-type saponins have been isolated from Panax ginseng C. A. Meyer, which vary in the number and type of monosaccharide residues present in the sugar side chains. The individual ginsenosides are named Rx according to their mobility on thin-layer chromatography plates. Examples of known ginsenosides include those in groups Ra through Rh. The isolation of three new dammarane-type saponins (named Rk1 to Rk3) from heat-processed ginseng has also been reported recently (see Park, I. H. (2002) Arch Pharm Res. 25: 428-32). No specific extraction process for extraction of the saponins has been disclosed, as emphasis is on use of the compounds for cancer treatment and not the extraction process. It has been indicated in the patent application that extracts can be prepared from plant material by standard techniques known in the art. The inventors cite a variety of strategies available for preparing extracts from plant material, the choice of which depends on the ability of the method to extract the components required for the compositions of the present invention. Examples of suitable methods for preparing extracts include, but are not limited to, hydro-distillation, direct steam distillation, solvent extraction, and Microwave Assisted Process. The steam and components can then be condensed and the extract collected. Organic solvents can also be used to extract the active components. Non-limiting examples of such organic solvents include methanol, ethanol, hexane, and methylene chloride. As an alternative to aqueous or organic solvents, microwaves can be used to excite water molecules in the plant tissue which causes cells to rupture and release the compounds trapped in the extracellular tissues of the plant material. To confirm the presence of the desired components in the extract (i.e. the saponins and/or sapogenins), a variety of analytical techniques well known to those of skill in the art may be employed. Such techniques include, for example, chromatographic separation of organic molecules (e.g., gas chromatography or liquid chromatography), mass spectroscopy, or other spectroscopic techniques (such as infra-red, ultra-violet or nuclear magnetic resonance spectroscopy).
Thus, the above patent application does not disclose a specific extraction process for the saponins of bioactive interest but only refers to existing conventional methods in general. Moreover, the saponins are altogether different from the saponins of the present invention which is specifically for steroidal saponins i.e. furostanolic saponins, particularly protodioscin. Also, the extraction process of saponins in the present invention is not a generalized scheme but is a specific novel process for the preparation of furostanolic saponin rich, bioactive extracts of high purity (>70%) from fenugreek seeds and has been described in detail.
8. Honeysuckle (Lonicera japonica Thunb.): United States Patent Application no. 20060014240 discloses an extraction and purification method of active constituents including sweroside from for honeysuckle stem (stem of honeysuckle where leaves are removed) by removing tannins, hardly soluble flavonoids, saponins, and the like. Thus obtained active constituents have better anti-inflammatory and analgesic effect, are safer and more stable than the conventional active constituents obtained from honeysuckle flower or honeysuckle leaves, and include sweroside which is an effective active ingredient of anti-inflammatory and analgesic drug. It discloses only the removal of saponins as a step for purification of the compounds of interest and does not disclose any specific process for purification of saponins at all.
9. Plant species, notably from the genera Smilax, Asparagus, Anemarrhena, Yucca and Agave: United States Patent Application no. 20060182817 discloses the use of a number of saponins and sapogenins, notably those of steroidal structure, in the treatment of cognitive disfunction and similar conditions. The saponins and sapogenins of principal interest in certain aspects of the present invention occur naturally in a range of plant species, notably from the genera Smilax, Asparagus, Anemarrhena, Yucca and Agave. The species presently of greatest interest include Smilax regelii Kilip & Morton—commonly known as Honduran sarsaparilla; Smilax aristolochiaefolia Miller—commonly known as Mexican sarsaparilla; Smilax omata Hooker—commonly known as Jamaican sarsaparilla; Smilax aspera—commonly known as Spanish sarsaparilla; Smilax glabra Roxburgh; Smilax febrifuga—Kunth—commonly known as Ecuadorian or Peruvian sarsaparilla; Anemarrhena asphodeloides Bunge; Yucca schidigera Roezl ex Ortgies; and Yucca brevifolia Engelm. Saponins and sapogenins which may be of interest also occur naturally in other genera, for example Dioscorea, Trillium, Solanum, Strophanthus, Digitalis and Trigonella. As indicated above, some saponins and sapogenins from these sources possess undesirable properties and are thus not recommended for use in the invention. Methods of treatment, and pharmaceutical compositions based on specific saponins/sapogenins are also disclosed. However, there is no disclosure at all about the extraction process for the saponins of interest.
10. Xanthoceras sorbifolia: United States Patent Application no. 20050220910 discloses compositions, methods and process of producing extracts from Xanthoceras sorbifolia. The extract comprises alkaloids, coumarins, saccharides, proteins, polysaccharides, glycosides, saponins, tannins, acid, flavonoids and others. The composition can be used for treating cancer, arthritis, rheumatism, poor circulation, arteriosclerosis and a wide variety of other ailments. The invention provides compounds comprising at least one sugar, a triterpene, such as Sapogenin, and at least one side chains at Carbon 21 and 22, such as Angeloyl groups. The structure of the compound has been described and has formula of C57H88O23 and the name 3-O-[.beta.-D-galactopyranosyl(1.fwdarw.2)]-.alpha.-L-arabinofuranosyl(1.-fwdarw.3)-.beta.-D-glucuronopyranosyl-21,22-O-diangeloyl-3.beta.,15.alpha.-16.alpha.,21.beta.,22.alpha., 28-hexahydroxyolean-12-ene, also known as Xanifolia-Y. This compound was isolated from Xanthoceras sorbifolia. 
The extraction process of the bioactive saponin has not been disclosed. Also, the bioactive saponin as disclosed in the above patent application (Molecular formula C57H88O23) is quite different from the bioactive saponin of the present invention, which is specifically a furostanolic saponin, protodioscin having molecular formula C51 H84O22.
11. Butter tree of the Sapotaceae family: United States Patent Application no. 20050233016 discloses a method for preparing a saponin-containing aqueous extract on the basis of a waste product from a butter tree of the Sapotaceae family, said method comprising the following steps: (i) mixing one part waste product with 4-30 parts of water; (ii) incubating the mixture formed in step (i) under alkaline conditions; and (iii) recovering an aqueous extract containing saponins by removing solids from the alkaline mixture formed in step (ii). The invention also relates to saponin-containing aqueous extracts obtainable by said method and uses thereof. Also, the saponins obtained are quite different from the saponins of the present invention. In the above patent, the extract obtained is composed of a diversity of water soluble types of constituents. One group is the tannins. The tannins are a group of simple and complex phenol, polyphenol and flavonoid compounds, bound with starches, and often they are just classified as tannins simply as they contain variations on gallic acid. One simple way of fractionating the constituents is by applying ultra filtration to separate the constituents according to their molecular size. Extracts obtained by the methods of the invention may be further subjected to a hydrolysing step converting a fraction of the saponins to their corresponding sapogenins. The sapogenin part may be further purified by recrystallisation and/or derivatised with fatty moieties to make it soluble in e.g. oil.
The extraction process as described in the above cited document is not restricted to a particular class of saponins, but is a very broad process encompassing a variety of water soluble compounds from waste by product of butter tree. Applications envisaged for the extracts include use as food additives, food ingredients, ingredients in detergent products; cosmetic product; in a pharmaceutical product for topical application; active component in a pharmaceutical product for lowering the level of serum cholesterol in a human being or in other mammals; as an active compound in a pharmaceutical product for treatment of inflammatory diseases; as an active compound in a pharmaceutical product for systemic administration, e.g. treatment of cardiac disorders or diuretic disorders, or vitamin D associated disorders; as an active component in the manufacture of a nutritional supplement; as an ingredient in immunostimulatory complexes (ISCOMs). Also, the plant extract e.g. may be used as a wetting agent or emulsifier. The extract can be used as a wetting agent in many applications e.g. spraying of pesticides and herbicides, dust control, etc. Furthermore it can be formulated with other surfactants, builders and ingredients normally used in detergents.
The process is quite different from the process of the present invention which is not based on aqueous extraction alone, but utilizes organic solvents and the process of gel filtration chromatography. Also, the saponins obtained are quite different from the saponins of the present invention. In the above patent, the extract obtained is composed of a diversity of water soluble types of constituents, whereas the process of the present invention discloses a specific method for extraction of furostanolic saponins as high purity fractions (>70%), from fenugreek seeds, only for pharmaceutical use and no other intended use.
Furostanolic Saponins
Furostanolic saponins (FUROSTANOLIC SAPONINS) are important phytochemicals. They are extracted from plants and are useful in the treatment of various diseases, as components of health foods and also as nutraceuticals. Furostanolic saponins have been shown in vertebrate studies to have positive effects on anabolic processes, testosterone production, appetite stimulation, and immune function. They are used for treating sexual impotency as well as for boosting muscle growth. Both of these effects stem from their ability to enhance testosterone levels in the body.
Current research indicates that the dominant furostanolic saponin, PROTODIOSCIN is the active ingredient responsible for testosterone-boosting effects. (CAS No. 55056-80-9; Molecular Formula C51H84O22). This steroidal saponin appears to stimulate the release of luteinizing hormone (LH) from the pituitary gland. LH then travels via the bloodstream to the testicles, where it stimulates testosterone production. Protodioscin is also believed to increase dehydroepiandrosterone (DHEA) production by the adrenal glands. This steroid precursor becomes testosterone.
Extraction of Saponins from Sources which are Regarded as Rich Sources of Furostanolic Saponins
Some of the commonly known plant sources regarded as rich sources of Furostanolic Saponins are Tribulus terrestris, Dioscorea deltoidea, Avena sativa and Fenugreek.    a) Tribulus terrestris: This herb is cultivated in many parts of the world, including the United States, Mexico and Asia. It it is used for treating sexual impotency, as well as for boosting muscle growth. Both of those effects stem from its ability to enhance testosterone levels in the body. Current research indicates that the dominant furostanolic saponin, protodioscin is the active ingredient in Tribulus extracts that affords it its testosterone-boosting effects.    b) Dioscorea deltoidea This climbing plant grown in Asia is used for numerous purposes-from treating asthma to killing lice. Its high concentration of furostanolic saponins makes it a good supplement for boosting testosterone levels in a manner similar to Tribulus terrestris and fenugreek.    c) Avena sativa: Avena sativa is the scientific name for oats. An extract from the plant has the ability to increase testosterone levels by enhancing LH release. Maybe that's why oatmeal is such a bodybuilding staple. A bowl of oatmeal probably doesn't have enough of the active compound avenacosides to give a noticeable boost, but concentrated Avena sativa extract does. Avenacosides are a type of plant saponin similar to furostanolic saponins.    d) Trigonella foenum-graecum (Fenugreek): The fenugreek herb is native to southern Europe, the Mediterranean region and western Asia. It has numerous health benefits, including increased libido, elevated testosterone and increased insulin release. As with Tribulus terrestris, this herb contains a high amount of furostanolic saponins (FUROSTANOLIC SAPONINS). Therefore, fenugreek extract works in a manner similar to Tribulus-elevating testosterone via increases in the amount of LH and DHEA the body produces. Some bodybuilders report having a better appetite when taking fenugreek extract-a great benefit for hardgainers.Extraction of Sponins from Tribulus 
United States Patent Application no. 20050163874 discloses a process for obtaining a pharmacological substance characterized by that it is a combination of bio-active trivalent chromium and a complex of steroid saponins obtained from the leaves, stems and fruit of the plant Tribulus Terrestris L. and consisting of furostanols, spirostanols, sapogenins, sterols, flavonoids and other biologically active substances typical of this plant. The pharmacological substance based on biologically active substances obtained from the plant Tribulus Terrestris L., characterized by processing an extract from the plant Tribulus Terrestris L. at negative temperatures until obtaining a complex of steroid saponins. Neither the extraction temperatures nor details of the solvents and conditions for extraction have been disclosed, though composition has been described in detail.
The complex contains furostanols, spirostanols, sapogenins, sterols, flavonoids and other biologically active substances typical of this plant in the following ratios in % by weight: Furostanols from 1 to 35%, Spirostanols from 1 to 40%, sapogenins, from 0.01 to 30% sterols from 0.01% to 15%, and flavonoids and other bio-active substances, from 0.0001 to 15%. The same is indicated to be used as an agent reducing blood sugar, improving blood circulation and especially blood circulation in veins and capillaries of the limbs of diabetic patients, reducing bad cholesterol level, increasing good cholesterol concentration, maintaining cardiovascular and liver functioning with an additional prophylactic or healing effect on the immune system and the immune resistance.
In contrast, the process of the present invention does not utilize extraction at negative temperatures nor does it involve addition of any metal such as chromium to the extracts. Moreover, the extraction process of the present invention is not directed towards extracting a mixture of various saponins as is given in the above patent of the prior art. Rather, it is directed only towards extracting one specific class of saponins viz. furostanolic saponins particularly protodioscin, present at a high purity level of >70% from seeds of fenugreek. No other parts of the plant are used. Also, the bioactive function of the saponins of the present invention is entirely different from that in the prior art document viz. elevation of testosterone levels, which is not mentioned at all in the prior art document.
United States Patent Application no. 20050208158 relates to a pharmacological composition based on biologically active substances obtained from leaves, stems and fruit of the plant Tribulus terrestris L. to be used as an agent strengthening the immune system, increasing T-cells and anti-bodies number and testosterone quantity in human organism and having a strong anti-virus and anti-inflammatory effect in the form of a medicine or as an addition to food.
The pharmacological composition represents a combination of bio-active zinc, bio-active selenium and a complex of steroid saponins obtained from leaves, stems and fruit of the plant Tribulus terrestris l. and consisting of furostanols spirostanols, sapogenins, sterols, flavonoids and other biologically active substances typical of this plant. The pharmacological substance based on biologically active substances obtained from the plant Tribulus Terrestris L., characterized by processing an extract from the plant Tribulus Terrestris L. at negative temperatures until obtaining a complex of steroid saponins. The complex of steroid saponins contains furostanols, spirostanols sapogenins, sterols, flavonoids and other biologically active substances typical of this plant in the following ratios in % by weight: furostanols from 1 to 90%, spirostanols from 1 to 75%, sapogenins, from 0.01 to 80%, sterols from 0.01% to 23%, flavonoids and other bio-active substances from 0.0001 to 24%. Pharmacological composition according to the invention is characterized in that the complex of steroid saponins obtained from the leaves, stems and fruit of the plant Tribulus Terrestris L., bio-active zinc and bio-active selenium are all included in a pharmaceutical or nutritive base in the following ratios in % weight: a complex of steroid saponins obtained from Tribulus Terrestris L. from 1 to 40%, bio-active zinc from 0.001 to 2%, pharmaceutical or a nutritive base from 1 to 55%.
The composition is to be used as an agent for strengthening the immune system, increasing T-cells and anti-bodies number and testosterone quantity in human organism and having a strong anti-virus and anti-inflammatory effect in the form of a medicine or as an addition to food.
However, neither the extraction temperatures nor details of the solvents and conditions for extraction have been disclosed, though composition has been described in detail. In contrast, the process of the present invention does not utilize extraction at negative temperatures nor does it involve addition of any metals such as zinc or selenium to the extracts. Moreover, the extraction process of the present invention is not directed towards extracting a mixture of various saponins. Rather, it is directed only towards extracting one specific class of saponins viz. furostanolic saponins particularly protodioscin, present at a high purity level of >70% from seeds of fenugreek. No other parts of the plant are used.
Extraction of Saponins from Dioscorea Deltoidea 
US patent application number 20070213280 describes a process for extraction of steroidal saponins (furostanolic saponins) from traditional Chinese medicine and natural products. Extraction procedures for two varieties of Discorea viz. D. nipponica and D. futschauensis have been described. The basic procedure in both is the same involving the steps of:                1. Extraction with alcohol by heating and refluxing        2. Suspending the extract in water to get a water soluble and water insoluble portion        3. Passing the extract through a resin column and eluting with water followed by increasing concentrations of ethanol viz. 10%, 50% and 95%.        4. Taking the 50% ethanol eluted fraction and subjecting it to silica gel column chromatography on column with granularity of 45.about.75 um, using a specific elution mixture of CH3Cl, CH3OH and H2O in the ratio of 8:2.5:0.1 and methanol, step by step        5. Vaporizing and concentrating the eluted solution under decreased pressure, incorporating the crystals of component fractions and re-crystallizing the crystals to get compounds of interest viz. MethylProtodioscin (MPD) and Pseudoprotodioscin (PPD).Extraction of Saponins from Fenugreek (Trigonella foenum-graecum)        
Fenugreek (Trigonella foenum-graecum) has attracted considerable interest as a natural source of soluble dietary fiber and diosgenin (sapogenins). The fenugreek seed contains a central hard, yellow embryo surrounded by a corneous and comparatively large layer of white, semi-transparent endosperm. This endosperm contains galactomannan gum. The endosperm is surrounded by a tenacious, dark brown husk. The color of the gum fraction depends upon the amount of outer husk (brown color) and cotyledon (yellow color) present. There are commercial uses for the various fractions of the fenugreek seed. The commercial fenugreek oleoresins are used as an ingredient for imitation maple flavors and is effective in butter, butterscotch, black walnut, nut and spice flavors. Another fraction of the fenugreek seed has been found to be a quantity of saponins.
Fenugreek seed saponins are steriodal in nature with diosgenin as the main sapogenin. Disogenin is used by the drug industry as a precursor to progesterone (steriod hormones) which is used in the manufacturing of oral contraceptives.
Processes for the extraction of bioactive fractions, not necessarily confined to saponins, from fenugreek seeds have also been described in the prior art. However, the bioactive fractions as described in the prior art are quite different from that of the present invention. None of the processes of the prior art reveals a process for the extraction of high-purity (>70%), bioactive furostanolic saponins fractions, particularly protodioscin, from fenugreek seeds.
Inventions of The Prior Art are Briefly Discussed Below:
U.S. Pat. No. 5,997,877 discloses a process for extraction of various useful fractions from fenugreek seeds (Trigonelle foenum-graecum). The process has a high yield rate, and provides a number of high-quality fractions of the fenugreek seed including a soluble dietary fiber fraction, de-flavored fenugreek seed, high-protein fenugreek meal, and dioscin and other saponins, along with the fenugreek oleoresins which have conventional commercial use. The main steroidal saponin extracted by the process of the invention is diosgenin, used by the drug industry as a precursor to progesterone (steriod hormones) which is used in the manufacturing of oral contraceptives. There is no disclosure about a processs, particularly for extraction of protodioscin rich furostanolic saponins, as has been disclosed in the present invention.
US patent application no. 20010024665 also discloses method for obtaining useful fraction i.e. substantially pure fixed oil(s), oleoresin and dietary fiber from Fenugreek seeds. The method employs two different solvent extraction stages, wherein the first extraction isolates fixed oils and the second extraction isolates oleoresin. The dietary fiber remaining after extraction is clean, approximately light yellow to light brown, substantially tasteless and substantially odorless.However, it does not disclose any process or method for extraction and purification of fursotanolic saponins as in the present invention.
US patent application no. 20040009247 discloses Fenugreek seed bio-active compositions for facilitating and supporting the metabolism and transport of glucose and carbohydrates into muscle cells and methods for extracting same. The bio-active compounds include 4-hydroxyisoleucine and one or more amino acids selected from the group consisting of arginine, aspartate, threonine, serine, glutamate, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tryptophan, phenylalanine, ornithine, lysine, histidine, gamma-amino butyrate, and trimethylhistidine. The composition of bio-active compounds includes between about 10% and 70% of 4-hydroxyisoleucine and between about 20% and 40% of the amino acids. Thus, the disclosure in the prior art is restricted to extraction of amino acid rich fractions only from fenugreek seeds. There is no disclosure at all regarding any extraction procedure for furostanolic saponins, particularly protodioscin rich fractions from fenugreek seeds as in the present invention.
United States Patent Application no. 20050153001 discloses a fenugreek seed extract to lower blood cholesterol. The extract is prepared fresh and involves merely boiling the soaked seeds of fenugreek in a cup of water for 5-7 minutes. There is no disclosure regarding the contents of the extract. In contrast, the extraction process of the present invention is a multi-step process involving use of various solvents, chromatrography etc. and is directed specifically towards the extraction of furostanolic saponins, particularly protodioscin.
Significance of the Present Invention
As discussed above, the three major sources of FUROSTANOLIC SAPONINS are Tribulus terrestris, Dioscorea deltoidea and Fenugreek. Various natural preparations of FUROSTANOLIC SAPONINS based on extracts of these are available commercially, but suffer from the limitation of variation in therapeutic efficacy or desirable results. One of the major reasons for this is the large variation in the content of the active compounds or the bioactive fractions. This results in variation of results, which affects the commercial value and sale of the products. Hence, it is highly desirable to have a commercially viable process which results in high concentration of the pharmacologically active compounds in the extracts. Theoritically, it is possible to keep on enriching the fractions to obtain high concentration of bioactive compounds, but this may end up making the process commercially unviable, resulting in non-availability of commercial preparations or the availability of preparations at affordable prices, but with low content of bioactive compounds i.e. FUROSTANOLIC SAPONINS.
An illustration for the same is as follows:
Tribulus terrestris has been sold in the sports nutrition industry for several years apparently as a means of raising levels of the hormone, testosterone. The main components of Tribulus responsible for this effect are FUROSTANOLIC SAPONINS. However, as per studies available, it was found that the concentration of the pharmacologically active components i.e FUROSTANOLIC SAPONINS was much less than the desirable limits. Available research demonstrates that a person should consume between 500 mg-2000 mg of these FUROSTANOLIC SAPONINS in order to consistently get the proper response. Thus, the bioactive fractions being sold should have sufficiently high percentage of the active biological compounds i.e. FUROSTANOLIC SAPONINS. Commercially available preparations of FUROSTANOLIC SAPONINS from tribulus have been reported to be containing only 20 mg of FUROSTANOLIC SAPONINS. This practically means that a person needs at least 25-50 capsules to get the desired results in terms of efficacy. Taking such a large number of capsules, on daily basis is not practically possible.
It has been reported (Derek Cornelius, 2008 www.qfac.com/articles/syntrax/tribulus.html) that none of the commercially available Tribulus preparations are standardized for furostanolic saponins. At best, they are standardized for saponins. The problem is that Tribulus contains a fairly large proportion of spirostanolic saponins. An even further problem is that many products actually contained 40% saponins but had less than 5% furostanolic saponins. Most U.S. testing labs only test for total saponins via a simple, crude testing method. Furostanolic saponins have specific biological properties while other saponins including the spirostanolic type have not been shown to exhibit the same potent effects. Hence, it is highly desirable to have preparations with high FUROSTANOLIC SAPONINS content.
Thus, there was a need for a natural preparation which would contain the biologically active FUROSTANOLIC SAPONINS in sufficienty large concentration, so that a comfortable dose of 1-2 capsules per day could be made possible. This worked out to be 70-75% of the biologically active FUROSTANOLIC SAPONINS in the extracts.
It was found by the inventors that the natural concentration of FUROSTANOLIC SAPONINS in Tribulus was too less and could not result in a commercially viable process. Thus, there was a need for identification of a suitably rich source of FUROSTANOLIC SAPONINS and standardize a commercially viable process for the same. The inventor identified two potentially good sources of FUROSTANOLIC SAPONINS viz. seeds of fenugreek (Trigonella foenumgraecum) and rhizomes of Singly Mingly (Dioscorea deltoidea) from which a commercially viable process could be developed, to extract the biologically active FUROSTANOLIC SAPONINS.
Out of these two, Fenugreek was the more suitable one owing to easy availability of seeds and low cost. Dioscorea deltoidea suffers from the limitation that that its rhizome portion is used, which is not as abundantly available as are fenugreek seeds and is an endangered species in some countries. Moreover, Fenugreek seeds are a cheaper raw material source and starting material as compared to rhizomes of Dioscorea deltoidea. 
A search of the Indian and International patent databases revealed that there was no process in the prior art for obtaining high-purity (>70%), bioactive, extracts of FUROSTANOLIC SAPONINS from Fenugreek seeds. Practical procedures for large scale quantitative and qualitative recovery of highly purified FUROSTANOLIC SAPONINS are lacking. The lack of suitable practical extraction and purification methods is also reflected in the relatively high cost of those extracts that are available.
Prior Art for Isolation of Furostanolic Saponins
The prior art for isolation of FUROSTANOLIC SAPONINS from plant materials broadly falls into two categories:    a) An aqueous extraction route: typically described by soaking and boiling the plant material in water resulting in the extraction of primarily water soluble compounds including saponins.
The disadvantages of the aqueous extraction method are:                Low yield: Percentage of desirable fraction obtained is quite low        Low purity: Purity of desirable fractions is very poor        Energy intensive: High temperatures are required for heating, which results in decomposition of the desired compounds.        Complicated process: Aqueous extraction of saponins i.e. ‘soapy compounds’ results in very high foaming which makes the overall operations complicated and difficult to control.            b) A hot organic solvent extraction process: characterized by use of polar solvents e.g. ethyl alcohol, methyl alcohol and butyl alcohol to extract compounds of interest.
The advantages of this process are:                High yield        High purity        Low energy requirements        Simple operations, as solvents are easy to evaporate which makes drying of the product fast; also there is no foaming        
Surprisingly, the inventors of the present invention, have determined that neither the aqueous extraction route nor the hot alcohol extraction route are particularly efficient in recovery of FUROSTANOLIC SAPONINS from fenugreek seeds, resulting in low recoveries and low purity fractions (3-5%). Thus, none of the approaches described or indicated in the prior art are practical for large scale production of high-purity (>70%), bioactive extracts of FUROSTANOLIC SAPONINS and are impractical for plants that contain mixtures of saponins.
A novel, highly efficient process from fenugreek seeds (Trigonella foenumgraecum) was developed by the inventors and same is disclosed in this application. The process results in high-purity (>70%), bioactive FUROSTANOLIC SAPONINS in which one of the bioactive fraction is protodioscin (>30%).